Detection of Serum HCV RNA by Rapid Cycle PCR Using Hot Air Thermocycler with Capillary Tubes.
- Author:
Mi Yae YOUN
1
;
Hye Soo LEE
;
Dal Sik KIM
;
Sam Im CHOI
;
Carl T WITTWER
Author Information
1. Department of Clinical Pathology, Chonbuk National University Medical School, Institute for Medical Science, Chonju, Korea.
- Publication Type:Original Article
- Keywords:
Hepatitis C Virus;
Rapid cycle PCR;
Hot air thermocycler;
Capillary tubes
- MeSH:
Capillaries*;
Diagnostic Tests, Routine;
Genome;
Glass;
Hepacivirus;
Hot Temperature;
Humans;
Plastics;
Polymerase Chain Reaction*;
RNA*
- From:Korean Journal of Clinical Microbiology
1999;2(1):89-94
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: Reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of Hepatitis C virus (HCV) RNA from serum. The PCR by conventional heat block thermocycler using small plastic tube is time and reagent consuming procedure, but rapid cycle PCR (RPCR) by hot air thermocycler using glass capillary tube is very rapid and economic. Therefore, RPCR have been recognized as a very convenient method for routine diagnostic test in clinical laboratories, but there are few reports about its usage for the detection of HCV RNA. METHODS: We selected two sets of primer pair from 5'noncoding region of HCV RNA genome, and optimized RPCR condition using hot air rapid thermocycler with master mix in capillary tubes. And RT-RPCR for detection of HCV RNA were performed on the serum of 58 patients, which were tested anti -HCV antibody by EIA. RESULTS: The optimized RPCR conditions were: denaturation; 94 degrees C for "0" sec, annealing; 55 degrees C (first) and 50 degrees C (nested) for "0" sec, elongation; 72 degrees C for "0" sec, and amplification cycles were 30 cycles. The consuming times per cycle were 30 sec (first) and 40 sec (nested), respecti vely, so the total involving times for nested RPCR were 35 min. Of the 42 EIA positive samples, 26 (62%) were RT-RPCR positive. CONCLUSIONS: RT-RPCR using hot air thermocycler with glass capillary tubes for detection of HCV RNA in serum is very rapid and economic than conventional PCR using heat block thermocycler. Therefore HCV RNA detection by RT-RPCR appears to be very useful for routine clinical laboratory diagnostic method.
